Protein tyrosine kinases are currently recognized as important molecular targets for drug development in the treatment of several disorders, particularly in the treatment of proliferative disorders. Dysregulation of tyrosine kinase activity has emerged as a major mechanism by which cancer cells evade normal physiological constraints on growth, proliferation and survival.
Tyrosine kinases (TKs) are enzymes that catalyze the transfer of phosphate from ATP to tyrosine residues in polypeptides. The human genome contains about 90 TK and 43 TK-like genes, the products of which control a wide variety of cellular events including cellular proliferation, survival, differentiation, function and motility.
TKs are divided into two main classes viz. receptor TKs and non-receptor TKs. Activities of both types of TKs are under tight control so that non-proliferating cells have very low levels of tyrosyl phosphorylated proteins. Receptor TKs become activated when ligand binds to the extracellular domain resulting in receptor oligomerization, disruption of the autoinhibitory juxtamembrane interaction and autophosphorylation of a regulatory tyrosine within the activation loop of the kinase. After activation, autophosphorylation generates binding sites for signaling proteins, recruiting them to the membrane and activating multiple signaling pathways.
The non-receptor TKs such as c-Abl, are maintained in an inactive state by cellular inhibitor proteins, lipids and through intramolecular autoinhibition. Non-receptor TKs are activated by diverse intracellular signals through dissociation of inhibitors, by recruitment to transmembrane receptors (causing oligomerization and autophosphorylation) and through transphosphorylation by other kinases. TK signaling is terminated in part through the action of tyrosine phosphatases that hydrolyze tyrosyl phosphates and by the induction of inhibitory molecules.
Dysregulation of TK activity arising out of mutation, over-expression or dysfunctional autoregulatory mechanisms has been implicated in many diseases including cancer. Given the multiple levels of regulation of TKs, it is not surprising that TKs are dysregulated in cancer cells by several ways. A common mechanism of TK activation in hematological cancers is the fusion of a receptor or non-receptor TK with a partner protein, usually as a consequence of a balanced chromosomal translocation. A primary example of this mechanism is Bcr-Abl, the non-receptor fusion TK in CML, in which a tetramerization domain in Bcr overcomes autoinhibition of Abl catalytic activity through oligomerization and autophosphorylation. With some receptor TKs, absence of the juxtamembrane inhibitory domain in the fusion protein contributes to activation. A second important mechanism of TK dysregulation is a mutation that disrupts autoregulation of the kinase. Mutations in the Fms-like tyrosine kinase 3 (FLT3) receptor in acute myeloid leukemia (AML) render this TK active in the absence of ligand; in another example, small deletions and point mutations in the kinase domain of epidermal growth factor receptor (EGFR) in a subset of non-small-cell lung cancers increase the sensitivity of the receptor to its ligand and alter receptor signaling. A third mechanism of TK dysregulation is increased or aberrant expression of a receptor TK, its ligand, or both. Examples include overexpression of the receptor TK ErbB2 (HER-2/neu) in breast cancer and overexpression of a mutant form of platelet-derived growth factor (PDGF), a receptor TK ligand, in dermatofibrosarcoma protuberans with t(11;17). Lastly, increased TK activity can result from a decrease in factors that limit TK activity, such as impaired tyrosine phosphatase activity or decreased expression of TK inhibitor proteins. Aberrant TK activation can increase the survival, proliferation and cytotoxic drug resistance of malignant cells; in tumors it can increase angiogenesis, invasiveness and metastatic potential.
The TK family of enzymes has emerged as an important class of targets for therapeutic intervention. TKs can be inhibited pharmacologically through multiple mechanisms. One of the key focus areas in anti-TK drug discovery is the design and development of small molecules that can directly inhibit the catalytic activity of the kinase by interfering with the binding of ATP or substrates. An important advantage of TK-directed therapy is the possibility to perform pharmacodynamic studies that correlate inhibition of the targeted TK in cancer cells with clinical responses to the drug.
The dysregulated TK in the hematological cancers is Bcr-Abl which has been implicated as the direct cause of CML. Imatinib mesylate (Gleevec®), a 2-phenylaminopyrimidine compound by virtue of its inhibition of several TKs viz. Abl, Abl-related gene product (ARG), c-Kit and PDGF receptor (PDGFR) has demonstrated remarkable clinical efficacy in CML. It induces complete hematological and cytogenetic remissions in most patients with chronic-phase, however is much less effective in the accelerated and blast-crisis phases of the disease. It is the first TK inhibitor to be approved as first line monotherapy and has revolutionized the treatment for CML. Recently it has been shown that imatinib mesylate prevents β-cell apoptosis under conditions of β-cell stress (PNAS, 2008, vol. 105, 18895-18900). This, together with the observation that improvements in type II diabetes has been noted in patients on imatinib therapy, leads to the hypothesis that kinase inhibitors may prove to be beneficial in the treatment of diabetes. The tyrosine kinase EGFR has been targeted with small molecule inhibitors such as Tarceva® and Iressa® for the treatment of patients with non-small cell lung carcinoma (NSCLC). Sutent® is approved for the treatment of certain tumors because of its multi-modal action on the tyrosine kinases including the vascular endothelial growth factor receptor (VEGFR), Kit and PDGFR. Inhibition of other kinases with small molecule inhibitors include the tyrosine kinase FLT3 that is expressed on blasts in most cases of acute myeloid leukemia (AML), the tyrosine kinases FGFR1, FGFR3, c-FMS, JAK and SYK in a range of malignant hematological disorders and ALK, c-Met and RET in a host of solid tumors.
Inhibiting TKs with ATP-competitive kinase inhibitors block the enzymatic activity of the kinases. Often treatment therapies result in drug resistance over a period. Quite often, drug resistance is largely on account of mutations that occur to prevent the pressures exerted by drug binding. Thus, despite success with Gleevec® to treat CML through inhibition of the oncogene Bcr-abl, clinical resistance to the drug has been observed. Of the multiple mechanisms of drug resistance, mutations of the Bcr-Abl kinase have been particularly problematic, with 50-90% of the resistance to Gleevec® arising from mutations in the kinase domain. Over 22 mutations have been reported to date, some of the most common being G250E, Q252H, Y253F/H, E255K/V, T315A/I, F317L/V, M351T, F359V and H396R.
The second generation agents such as nilotinib (Tasigna®) and dasatinib (Sprycel®) are able to inhibit a large number of clinically relevant mutations. However, neither of these inhibit the T315I mutation (also known as the gatekeeper mutation), although this mutation is the largest singly occurring mutation to the current standard of care for CML viz. Gleevec®. Mutation of the gatekeeper residue enables the protein to bind ATP and continue to function. At the same time, Gleevec® is selectively rejected since it makes use of a hydrophobic pocket close to the ATP binding site, which ATP does not utilize. In fact, almost all small molecule inhibitors that are ATP-competitive utilize this hydrophobic pocket to attain much higher potency over ATP, Gleevec® is no exception. It is therefore not surprising that the gatekeeper and its mutation across numerous kinases are well known since most small molecule inhibitors of kinases are ATP competitive.
The Src family which consists of at least eight members (Src, Fyn, Lyn, Yes, Lck, Fgr, Hck and Blk) that participate in a variety of signaling pathways represents the major family of cytoplasmic protein tyrosine kinases. The prototypical member of this tyrosine kinase family is Src, which is involved in proliferation and migration responses in many cell types. Src activity has been shown to be elevated in different cancers, e.g. breast, colon, pancreatic and liver tumors. Highly increased Src activity is also associated with metastasis and poor prognosis. Antisense Src message impedes growth of colon tumor cells in nude mice, suggesting that Src inhibitors could slow tumor growth. Furthermore, in addition to its role in cell proliferation, Src also acts in stress response pathways, including the hypoxia response.
In addition, Src family kinases such as Lyn and Src are important in the Fc epsilon receptor induced degranulation of mast cells and basophils that plays an important role in asthma, allergic rhinitis and other allergic disease.
The lymphocyte-specific kinase (Lck), belonging to the Src family of tyrosine kinases, is expressed in T cells and natural killer (NK) cells and is responsible for the activation of and signaling through the T-cell receptor. This activation cascade results in the upregulation of inflammatory cytokines such as IL-2 and interferon (IFN)-γ, and ultimately in the activation and proliferation of T lymphocytes to generate an immune response. Inhibition of Lck is therefore likely to elicit an immunosuppressive effect that could be useful in the treatment of T-cell-mediated diseases like rheumatoid arthritis, inflammatory bowel disease, psoriasis, and organ graft rejection.
Classical tyrosine kinase inhibitors, which are predominantly the Bcr-Abl kinase inhibitors that are currently in clinical use, are described in the following patent literature:                U.S. Pat. No. 5,521,184 (the '184 patent): Exemplifies 4-[(Methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamide methanesulfonate (Imatinib mesylate, Gleevec®)        U.S. Pat. No. 7,169,791 (the '791 patent): Exemplifies 4-Methyl-N-[3-(4-methyl-imidazol-1-yl)-5-trifluoromethyl-phenyl]-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-benzamide (Nilotinib, Tasigna®)        U.S. Pat. No. 6,596,746 (the '746 patent): Exemplifies N-(2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide (Dasatinib, Sprycel®)        
While the second generation TK inhibitors in clinic viz. nilotinib and dasatinib have provided additional treatment option to patients who have developed resistance to imatinib, there are certain shortcomings with regard to their side effects. Particularly in the case of dasatinib, the increased potency may be associated with untoward off-target toxicities, which probably relate to their inhibitory activity against a broader range of protein kinases such as Kit, PDGFR and ephrin receptor (EphA2) tyrosine kinases which are directly implicated in haematopoiesis, control of tissue interstitial-fluid pressure and angiogenesis. These effects may provide the physiological explanation for some of the toxicities associated with dasatinib therapy such as myelosuppression and pleural effusion. Besides, treatment with highly potent Abl kinase inhibition has potential for the development of cardiotoxicity in patients with CML.
Although the second generation TK inhibitors in clinic provide treatment alternatives for patients who develop resistance to imatinib therapy, the prognosis for the patients having T315I mutation is not good since none of these currently marketed therapies are effective. There is thus an unmet medical need with regard to treatment of patients having the T315I mutation. Omacetaxine (homoharringtonine) is currently being evaluated by the FDA for CML patients with T315I. However, it is an intravenous drug with a non-specific mechanism of action. Other drug candidates in clinical phase include the Deciphera compound DCC-2036 (PCT Publication No. WO 2008/046003) and the Ariad compound AP24534 (Ponatinib, PCT Publication No. WO 2007/075869).
There is thus a need for newer selective TK inhibitors which are orally active, safer than existing therapies particularly with regard to decrease in cardiac toxicity associated with hERG/QT prolongation, and efficacious against the kinase mutations, including the T315I mutant for which there is currently no approved therapy. The current invention describes novel diarylacetylene hydrazide containing compounds which are potent inhibitors of Abl tyrosine kinase and their mutated forms, including the T315I mutant.